Equation 5: Single (a) and double (b) proton titration equation
5. Summary and Conclusion
Thiamine diphosphate (ThDP) dependent enzymes play central roles in the metabolism of biological systems and these enzymes possess powerful ability to catalyse the formation and breakage of carbon-carbon bonds. A variety of reactions are involved in this cofactor mediated enzymatic reactions such as decarboxylation (in pyruvate decarboxylase (PDC)), carboligation (in transketolase (TK) or acetolactate synthase (AHAS)) and oxidative transformations (in pyruvate dehydrogenase complex (PDHc) or pyruvate oxidase (POX)) (Kluger and Tittmann, 2008). The catalytic mechanism of ThDP-dependent enzymes has been extensively studied over the last 50 years after Breslow (Breslow, 1957) discovered in 1957 that the deprotonation at the C2 of the thiazolium ring is the first reaction step. Among these enzymes, transketolase is a very representative example which catalyses the reversible transfer of a C2-unit from ketose phosphates to the C1 position of aldose phosphates (Mitschke et al., 2010).
Previous colleagues in our group have solved the crystal structure of the genius covalent intermediates at ultra-high resolution and characterised a scissile bond elongation and out-of-plane distortion to be essential in the catalytic process of TK-reactions. This doctoral thesis is a follow-up work based on these extraordinary findings and presents a more comprehensive study of transketolase which involves the participation of a low barrier hydrogen bond (LBHB). As illustrated before, a LBHB is a special type of hydrogen bond when the pKa values of the H-bond donor and acceptor are matched. This leads to the formation of a short and strong hydrogen bond (bond length is around 2.55 Å). After careful reanalysis of the previously obtained sub-angstrom resolution crystal structures, a LBHB with a distance of 2.56 Å is unexpectedly observed between a conserved glutamate (Glu366 in hTK) and a neighbouring glutamate (Glu160) from the adjacent subunit. A hydrogen atom is unambiguously modelled almost at the equidistant position between the H-bond donor and acceptor. More interestingly, the observed LBHB locates in a proton tunnel which can synchronize the two active sites by several acidic amino acid residues and water molecules.
This proton wire was first proposed in the E1 component of pyruvate dehydrogenase complex (PDHc) and received wide acceptance among ThDP-dependent enzyme family.
In order to investigate the catalytic function of the LBHB in hTK, a single mutation variant E160Q was generated and the crystal structure with donor substrate F6P was determined at ultra-high resolution. Instead of the LBHB, an ordinary hydrogen bond (bond length around 2.80 Å) is observed with a well-defined protonation state for the amide function. The crystal structure of this variant at ground state also reveals the existence of an ordinary hydrogen bond, although the protons on the amide group can’t be precisely determined. Furthermore,
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the F6P-ThDP covalent intermediate in the crystal structure of hTK-E160Q exhibits identical scissile bond elongation and out-of-plane distortion as that in the wild type, suggesting a similar energy profile for both proteins. Steady-state kinetic analysis reveals that the enzymatic activity (kcat) of the wild type (2.79 ± 0.06 s-) is five times faster than the E160Q mutant (0.54
± 0.01 s-), but both proteins exhibit similar substrate affinity for the donor substrate X5P (Km
for wild type and E160Q is 73.9 ± 6.7 mM and 78.0 ± 4.8 mM, respectively). Pre-steady-state kinetic analysis by stopped-flow technique revealed that the donor half reaction of hTK wild type exhibits positive cooperativity (n = 1.56 ± 0.26 for AP depletion and 1.49 ± 0.43 for IP formation) and is two times faster than that of the E160Q variant which shows no cooperativity (kobsmax of wild type and E160Q for AP depletion is 9.06 ± 0.72 s- and 5.91 ± 0.43 s-, respectively). Those structural and kinetic results indicate that the LBHB in hTK plays a role in enzymatic efficiency and positive cooperativity for the two active sites. A Circular Dichroism (CD) based pH titration experiment reveals the pKa difference between human TK wild type and E160Q for the [AP] + [IP])/[APH+] equilibrium is around 1.5 pH units which corresponds to 2.01 ± 0.01 kcal/mol. This experimental result would revise the current knowledge about the energy profile of LBHBs which is obtained from computational calculation.
In the next part of this thesis, crystal structures of hTK with non-phosphorylated sugars xylulose and fructose lead to the accumulation of the post-cleavage DHEThDP intermediate, which is further confirmed by the H1-NMR and UV-Vis spectroscopy. The leaving groups D-glyceraldehyde and D-erythrose are also captured within the crystal structures and are found at the entrance of the active site. Stopped-flow analysis of the non-phosphorylated sugars with hTK reveals a very slow reaction process. The depletion of AP form, the formation of IP and enamine form have shown identical reaction velocity at the same substrate concentrations, indicating a same molecular origin for those different forms. Five-carbon sugar xylulose shows a 15-fold increased reaction velocity relative to the six-carbon counterpart fructose. This is consistent with the literature reported conformational equilibrium compositions of sugars that xylulose has 20 % active keto form while fructose only shows 0.5 %. The reactivity of the accumulated DHEThDP intermediate is confirmed by a sequential stopped-flow experiment using the native substrate R5P as the acceptor. Positive cooperativity (n = 1.67 ± 0.27) is observed for the acceptor half reaction with Vmax of 92.3 ± 6.1 s- and Ksapp of 0.71 ± 0.14 mM.
Supplement of exogenous phosphite dianion to human TK is shown to have activation function for the donor half reaction using the non-phosphorylated sugars. A 2-3 fold reaction acceleration of saturation phosphite concentration is observed for both 5-carbon and 6-carbon sugars. X-ray crystal structure in complex with phosphite dianion shows an induced
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conformation change of Ser345 which potentially lowers the binding energy thus enhances the reaction. In addition, the phosphite dianion is bound at a remote but same position where phosphate group of native substrates binds, revealing the potential role for the native substrates’ phosphate group.
In the last part of this thesis, intrinsic dynamics of several catalytic residues have been studied.
Ring flipping of a histidine side chain is observed in E.coli transketolase and several related enzyme mutants are studied. In human TK, a structural plasticity of the active site has been observed and site-directed mutagenesis studies give a preliminary insight into this intrinsic dynamics.
In general, this doctoral thesis has presented a comprehensive study of a LBHB in transketolase and have characterised a DHEThDP intermediate obtained from non-phosphorylated sugars. Those results indicate that although ThDP-dependent transketolase has been extensively studied over the last 30 years, a lot of knowledge for this enzyme as well as the whole ThDP-dependent enzyme family are still missing. More work from both experimental and computational aspects should be conducted in the future.
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6. Appendix
Table 6: X-ray statistics for the crystal structures of human transketolase E160Q ground state, E160Q in covalent complex with donor substrate fructose 6-phosphate (F6P) and E366Q in non-covalent complex with donor substrate xylulose 5-phosphate (X5P).
hTK-E160Q
Wavelength (Å) 0.97625 0.97625 0.97625
Space group C2 C2 C2 No. of reflections 1692285 (280303) 1383209 (232616) 304158 (53182)
No. of unique
reflections 263585 (47044) 238255 (39930) 90973 (15949) Completeness (%) 97.3 (93.5) 96.4 (94.4) 97.7 (97.8)
I/sigma (I) 14.73 (2.00) 13.94 (2.43) 31.48 (2.44)
Rmeas (%) 5.8 (94.6) 5.7 (74.3) 2.1 (74.5)
Redundancy 6.42 (5.96) 5.81 (5.83) 3.3 (3.3)
CC1/2 (%) 99.9 (72.7) 99.9 (94.4) 100 (79.6)
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hTK-E160Q ground state
hTK-E160Q + F6P
hTK-E366Q + X5P Refinement
Resolution range (Å) 50 - 1.04 25 - 1.08 25 - 1.5
Rwork/Rfree (%) 11.26/13.14 11.12/13.05 15.83/18.99 Number of atoms
Protein 5748 5860 4834
Ligands 14 95 51
Water 681 670 436
B-factor Protein 14.64 15.31 39.30
B-factor Ligands 22.33 26.14 38.80
B-factor Water 27.31 27.95 45.70
Deviations from ideals (r.m.s.d.)
Bond distances (Å) 0.010 0.011 0.011
Bond angles (°) 1.476 1.83 1.38
Dihedrals (°) 10.53 11.25 13.30
Ramachandran plot Favoured regions
(%) 98 98 98
Allowed regions (%) 2 2 2
Outlier regions (%) 0 0 0
B-factor from Wilson
Plot (A2) 10.74 11.37 27.08
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Table 7: X-ray statistics for the crystal structures of human transketolase wild type in complex with non-phosphorylated sugars xylulose and fructose as well as human transketolase wild type with activating phosphite dianion. No. of reflections 1251117 (182855) 597042 (105119) 313356 (94543)
No. of unique
reflections 223490 (31820) 342211 (59465) 65740 (19699) Completeness (%) 98.9 (98.1) 94.2 (92.9) 97.4 (97.7)
I/sigma (I) 16.96 (2.53) 5.29 (1.38) 28.14 (2.70)
Rmeas (%) 6.6 (91.1) 10.8 (65.1) 2.5 (67.1)
Redundancy 6.8 (6.7) 1.7 (1.7) 3.3 (3.2)
CC1/2 (%) 99.9 (81.5) 99.2 (61.5) 99.9 (75.2)
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hTK-wt + xylulose
hTK-wt + fructose
hTK-wt + phosphite Refinement
Resolution range (Å) 50 - 1.5 25 - 1.5 25 - 1.48
Rwork/Rfree (%) 14.42/17.76 15.69/18.10 15.04/17.53 Number of atoms
Protein 11021 10652 5320
Ligands 132 120 53
Water 1130 1232 632
B-factor Protein 16.25 18.32 17.52
B-factor Ligands 18.35 19.63 08.98
B-factor Water 20.35 22.12 22.56
Deviations from ideals (r.m.s.d.)
Bond distances (Å) 0.016 0.009 0.006
Bond angles (°) 1.648 1.281 1.101
Dihedrals (°) 13.604 12.909 12.930
Ramachandran plot Favoured regions
(%) 97.39 97.16 97.82
Allowed regions (%) 2.45 2.68 2.02
Outlier regions (%) 0.15 0.15 0.16
B-factor from Wilson
Plot (A2) 28.65 23.68 29.86
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Table 8: X-ray statistics for the crystal structures of human transketolase active site variants Q428A, S427A and H77A in complex with donor substrate fructose 6-phosphate (F6P).
hTK-Q428A No. of reflections 520841 (330481) 1456399 (312845) 142956 (86063)
No. of unique
reflections 85872 (55563) 336472 (74724) 19955 (12111) Completeness (%) 91.4 (87.3) 99.3 (99.0) 95.4 (94.2)
I/sigma (I) 7.0 (1.7) 15.04 (2.29) 3.95 (0.95)
Rmeas (%) 9.0 (75.2) 5.3 (79.1) 20.6 (101.9)
Redundancy 1.8 (1.8) 2.3 (2.2) 1.8 (1.7)
CC1/2 (%) 99.7 (75.2) 100 (81.8) 97.8 (25.2)
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hTK-Q428A + F6P hTK-S427A + F6P hTK-H77A + F6P
Refinement
Resolution range (Å) 50 - 1.5 50 - 1.25 50 - 1.9
Rwork/Rfree (%) 16.89/19.24 13.46/16.23 17.36/22.68 Number of atoms
Protein 10065 10135 5320
Ligands 120 132 52
Water 998 1032 556
B-factor Protein 21.35 20.12 45.65
B-factor Ligands 23.25 21.22 43.02
B-factor Water 30.98 30.21 48.74
Deviations from ideals (r.m.s.d.)
Bond distances (Å) 0.008 0.010 0.010
Bond angles (°) 1.628 1.783 1.605
Dihedrals (°) 13.963 14.101 14.525
Ramachandran plot Favoured regions
(%) 97.32 97.39 97.33
Allowed regions (%) 2.53 2.45 2.52
Outlier regions (%) 0.15 0.15 0.15
B-factor from Wilson
Plot (A2) 28.21 23.25 40.21
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Table 9: X-ray statistics for the crystal structures of E.coli transketolase active site variants D469N, D469A holo-enzyme in ground state and D469N apo-enzyme.
EcTK-D469N EcTK-D469A EcTK-D469N (apo) Data collection
Wavelength (Å) 0.91 0.82656 0.91
Space group P212121 P212121 P212121
Cell dimensions
a (Å) 89.9 90.0 90.5
b (Å) 102.2 101.7 101.7
c (Å) 133.1 133.1 133.2
α (°) 90.0 90.0 90.0
β (°) 90.0 90.0 90.0
γ (°) 90.0 90.0 90.0
Resolution range (Å) 25 - 1.06 (1.10 - 1.06)
50 - 1.15 (1.20 - 1.15)
25 - 1.2 (1.2 - 1.1) No. of reflections 6073900 (1382988) 1862797 (425446) 10349386 (1181962) No. of unique
reflections 232454 (54163) 207057 (49648) 1819659 (214657) Completeness (%) 95.7 (92.5) 99.0 (97.0) 98.1 (97.3)
I/sigma (I) 8.98 (2.81) 15.99 (3.40) 11.36 (4.48)
Rmeas (%) 10.2 (39.7) 6.0 (47.4) 31.6 (60.7)
Redundancy 4.3 (4.2) 5.2 (5.1) 8.5 (8.3)
CC1/2 (%) 99.8 (77.9) 99.9 (87.5) 90.5 (73.3)
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EcTK-D469N EcTK-D469A EcTK-D469N (apo) Refinement
Resolution range (Å) 25 - 1.06 50 - 1.15 25 - 1.2
Rwork/Rfree (%) 11.24/12.57 14.06/15.81 13.16/15.35 Number of atoms
Protein 12301 12166 10453
Ligands 243 210 65
Water 1862 1601 1657
B-factor Protein 10.65 16.63 n.d.
B-factor Ligands 14.32 23.21 n.d.
B-factor Water 20.24 25.98 n.d.
Deviations from ideals (r.m.s.d.)
Bond distances (Å) 0.011 0.007 0.010
Bond angles (°) 1.534 1.357 1.425
Dihedrals (°) 14.530 14.338 13.226
Ramachandran plot Favoured regions
(%) 98.40 98.33 98.55
Allowed regions (%) 1.60 1.67 1.45
Outlier regions (%) 0.00 0.00 0.00
B-factor from Wilson
Plot (A2) 10.35 20.74 26.32
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Fig 59: A possible low barrier hydrogen bond (LBHB) observed in the crystal structure of EcTK-D469N at a resolution of 1.06 Å. A LBHB is observed between Glu160 and Glu411 from neighbouring subunit with a hydrogen bond distance about 2.56 Å. The aminopyrimidine ring of ThDP, Glu160 and Glu411 are shown as ball and stick representation and surrounded by the calculated 2mFo-DFc map (contoured at 4σ level, blue). A hydrogen atom shown as sphere in grey could be observed at nearly equidistant position between E411 and E160 and is surrounded by a mFo-DFc map (contoured at 3σ level, green). An ordinary hydrogen bond with distance of 2.66 Å between N1’ and E411 is also shown for comparison.
Fig 60: Temperature jump analysis of human TK E160Q. The AP tautomeric form of ThDP on hTK-E160Q (5 mg/ml in 50 mM glycylglycine, 5 mM CaCl2, pH 7.6) was studied by using the temperature jump relaxation technique with a jump of 10 oC (12 to 22 oC) at 325 nm. a.) Progress curve of 50 individual traces in one measurement. Please note that the increasing UV absorbance is caused by the precipitation of protein. The average of those 50 traces is shown in red. b.) Comparison of human TK-E160Q with wild type. The blue curve indicates that communication between the two active sites of transketolase is interrupted by the mutation.
148
Fig 61: Steady-state kinetic analysis of human TK wild type and LBHB-related active site variants. a.) Progress curve of human TK wild type. The concentrations of X5P (in mM) for each curve are shown. The concentration of R5P is kept at 5 mM for all the measurements. b.) Progress curve of human TK E160Q. c.) Progress curve of human TK E160A. d.) Kinetic plot of the steady-state activity for hTK E160Q. Blue curve represents the fitting according to the Michaelis-Menten equation while the red curve represents the fitting according to the substrate inhibition equation. e.) Kinetic plot of the steady-state activity for hTK E160A. f.) Kinetic plot of the steady-state activity for hTK E165Q.
149
Fig 62: Detailed view of the carbanion-enamine intermediate trapped in hTK by using fructose at a resolution of 1.5 Å. a). The DHEThDP intermediate is shown in ball-stick representation with the 2Fo-Fc map contoured at 1σ level (blue) shown for ThDP moiety and the Fo-Fc map contoured at 3σ level (green) shown for dihydroxyethyl moiety of DHEThDP intermediate. b). Close-up of the intermediate that highlights the deviations from planarity of the thiazolium moiety (top) and aminopyrimidine moiety (bottom) by shown with auxiliary planes. Selected atoms and bond lengths are labelled.
150
Fig 63: Photodiode array based stopped-flow analysis of human transketolase with non-phosphorylated sugar fructose. Single-mixing stopped-flow analysis (10 mm path length) for the reaction of 5 mg/ml hTK, 5 mM CaCl2, 50 mM glycylglycine (pH 7.6) with 50 mM fructose at 4 °C and measured by photodiode array. a.) Progress curve for the reaction after 4 s and 2000 s after mixing.
b.) The difference spectra of the reaction after 2000 s and 4 s. Please note that the huge absorbance increase at 270-280 nm indicates the accumulation of erythrose in the reaction mixture. Inset: zoom-in view of the spectra indicates the decrease of AP form and the increase of enamine form. c.) UV-Vis spectra of standard erythrose with strong maximal absorbance at 260-270 nm. d.) UV-Vis spectra of standard fructose with very small absorbance at 260-270 nm compared with erythrose.
151
Fig 64: Single-mixing stopped-flow analysis of human TK with fructose. Plotting of absorbance amplitude against utilised fructose concentration reveals an apparent Kd value of 36.2 ± 3.7 mM.
Reaction condition refers to Fig 39.
Fig 65: Single-mixing stopped-flow analysis of E.coli TK with fructose. EcTK (4 mg/ml) was first supplemented with 300 µM ThDP and incubated for 20 min to reconstitute the holo-enzyme, then mixed with increasing concentrations of fructose in 5 mM CaCl2, 50 mM glycylglycine (pH 7.6) and measured by a single-mixing stopped-flow at 322 nm at 4 °C. a.) Representative stopped-flow reaction progress curve with 100 mM fructose. A biphasic process is observed compared to human TK. b.) Plotting of the reaction velocity of AP depletion (322 nm) against utilised fructose concentration. Please note that at same substrate concentration, the rate constants of E.coli TK is 40 times faster than that of the human TK.
152
Fig 66: Circular Dichroism (CD) spectra of substrate binding to human and E.coli TK. Non-phosphorylated substrates xylulose and fructose, native substrate F6P and artificial substrate HPA were added to human and E.coli transketolase and the binding behaviours were measured by CD. For experimental details, please refer to Table 2. a.) The binding of xylulose to human TK. b.) The binding of fructose to human TK. c.) The binding of fructose 6-phosphate (F6P) to human TK. d.) The binding of hydroxypyruvate (HPA) to EcTK-D469N. Please note that the absorbance at 300 nm drops after 20 min incubation, indicating instability of the DHEThDP intermediate.
153
Fig 67: “Flipping histidine” observed in the active site of EcTK D469N apo-enzyme. a.) Active site architecture of EcTK D469N-apo at a resolution of 1.20 Å. Amino acid residues His66, His100, His473 and Asn469 are shown in ball-stick representation surrounded by a 2mFo-DFc map (contoured at 2σ level, blue). The electron density for His473 is more diffuse while others (His66 and His100) are well-defined. Several water molecules are observed at the position where cofactor ThDP should bind. Water molecules are shown as red spheres and hydrogen bonding interactions are shown as black dashed lines.
b.) Anisotropic motion of His66, His100 and His473 in EcTK D469N apo-enzyme. The atomic displacement parameters (ADPs) of His473 indicate that Nε and Cε would thermally move along their bond axis, which is not possible because of rigid bond rule. This suggests that His473 exists in two conformation though ring flipping.
154
Fig 68: Single-mixing stopped-flow analysis of EcTK-D469N and EcTK-D469A with physiological substrate fructose 6-phosphate (F6P). EcTK (4 mg/ml) was first supplemented with 300 µM ThDP and incubated for 20 min to reconstitute the holo-enzyme, then mixed with increasing concentrations of F6P in 5 mM CaCl2, 50 mM glycylglycine (pH 7.6) and measured by a single-mixing stopped-flow at 320 nm at 25 °C. a.) Representative stopped-flow reaction progress curve of EcTK-D469N. a.) Representative stopped-flow reaction progress curve of EcTK-D469A.
Fig 69: Purification of hTK for crystallization. In order to obtain hTK with high purity for crystallization purposes, a Superdex 200 (S200) column was used to separate the protein samples after Ni-NTA purification. a.) Typical chromatogram of the purification step using the S200 column. b.) SDS-PAGE analysis of the fractions eluted from S200 column. Please note that the protein samples from the second peak is more pure than the first one therefore was used in the crystallization experiments.
155
Table 10: Analysis of the hydrogen bond distance at the same LBHB position in other transketolase structures. Transketolase structures with a resolution higher than 2 Å are analysed.
Organism Enzyme stage Distance (Å) PDB code Resolution (Å) H. sapiens
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